基于共培养策略挖掘一株海洋放线菌的聚酮类化合物及其防污活性研究

It is one of the most important strategies that the search for effective, and environment- friendly antifouling agents from marine natural products. In our previous work, totally 23 polyketide compounds including 11 new compounds, were isolated from a marine-derived actinomycete, Streptomyces sp．SCSGAA0027. All of these compounds were tested for antifouling activity and three known compounds of them showed evident effect. The whole-genome sequencing for the actinomycete was also performed, which indicated it encompass 13 modules of a type I PKS. Among them, only five PKS genes expressed under standard laboratory growth conditions, while the other eight ones were silent. During the co-culturing experiments with the actinomyces Streptomyces sp．SCSGAA0027 and each of more than 40 various fungal strains, three fungal strains were observed to elicit different secondary metabolites which are not produced in the actinomyces in single culture. Now this project will try to demonstrate the cryptic new antifouling polyketides from the actinomycete Streptomyces sp．SCSGAA0027 by a co-culture based approach toward activation of polyketide production from three of actinomyces-fungus co-cultures above. In this study, some work will be performed as follows. 1) Three of co-cultures are used for large-scale cultivations, respectively, affording their corresponding crude extracts. 2) Some new polyketides are discovered by separation of the extracts on repeated column chromatography (CC) under activity guidance. 3) The complete structures of new polyketides are determined by spectral analysis and chemical derivatizations. 4) The bioactivity of the isolated compounds is evaluated toward barnacle Balanus amphitrite and Bugula neritina larvae for antifouling and Shewanella oneidensis 817-9 for anti-biofilm. 5) The structure-function relationships for the active lead compounds are also investigated, generating an optimized molecular structure with additional characters of lower-toxicity and higher effectivity. 6) Several active lead compounds collected in a large quantity will be used as paint coating on a panel and tested in a marine mesocosm.

海洋天然产物是开发高效、无毒新型海洋防污剂的重要来源之一。申请人前期从一株海洋放线菌Streptomyces sp．SCSGAA0027的次级代谢产物中获得23个聚酮类化合物，包括11个新化合物和3个抗污损活性较显著的已知化合物，进一步的全基因组扫描和基因簇分析发现该菌株的13个PKS基因簇中仅表达了5个，还有8个沉默基因簇尚未表达。本项目拟在前期的工作基础上，利用共培养的方法激活放线菌Streptomyces sp．SCSGAA0027沉默基因的表达，运用现代天然产物的分离技术和波谱分析手段，结合抑制海洋污损生物草苔虫幼虫和藤壶金星幼虫附着以及海洋希瓦氏菌生物被膜形成两种活性筛选模型，深入挖掘放线菌潜在的新结构聚酮类化合物及其抗污损活性，探讨活性化合物的构效关系，大量制备或合成活性先导化合物并进行野外海上挂板现场测试，旨在发现具有原创性和自主知识产权的海洋防污剂。

海洋生物污损每年给全球的经济损失达近百亿美元，传统的有机防污涂料有机锡（TBT）和氧化亚铜（Cu2O）等尽管具有较好的防污效果，但由于破坏生态环境已被全球陆续禁止使用。当前市场上也还没有高效、环保的新型海洋防污剂作为替代品。海洋天然产物活性线显著且易降解，是开发高效、无毒新型海洋防污剂的重要途径之一。在前期，我们从一株海洋放线菌Streptomyces sp．SCSGAA0027的次级代谢产物中获得20余个聚酮类化合物。随后，在本项目资助下，我们基于共培养策略深入挖掘这株放线菌的聚酮类化合物，结合典型海洋污损大型生物草苔虫幼虫和藤壶金星幼虫防污活性以及海洋污损细菌解淀粉芽孢杆菌生物被膜形成抑制活性两种活性筛选模型，筛选出防污活性单体化合物。项目取得了以下成果，共分离鉴定45个化合物，包括14个新化合物；筛选出了2个防污活性较显著的聚酮化合物herbimycin C 和oxohygrolidin，IC50值分别达到2 µg/mL，5.3 µg/mL ，筛选出了1个显著抑制海洋细菌解淀粉芽孢杆菌生物膜形成活性的明星安沙霉素分子hygrocin C，IC50值达到3 µg/mL，herbimycin C 在野外挂板实际实验中表现出较好的防污效果，本项目的研究丰富了海洋天然产物结构的种类，也为聚酮类防污活性海洋天然产物的潜在应用和开发提供借鉴意义。